Below is a Google-cached version of Protein NMR - A Practical Guide - Solution NMR Assignment Theory page from www.protein-nmr.org.uk crashed on 11/6/11. You may want to check first if the site has been restored.
Assignment - Theory
The most simple and straight forward method of backbone resonance assignment involves the use of 15N,13C labelled protein and the measurement of
CBCANNH and
CBCA(CO)NNH spectra.
Large Proteins
Large proteins give worse NMR spectra, because they tumble more slowly. For this reason the CBCANNH and CBCA(CO)NNH spectra of larger proteins (> 150 residues) are often not of sufficient quality to be able to carry out a full assignment. In this case a good option is the use of
HNCA,
HN(CO)CA,
HNCO and
HN(CA)CO spectra.
Deuteration
For very large proteins (typically > 250 residues) it may become necessary to deuterate the protein. In this case varients of the CBCANNH and CBCA(CO)NNH are used (so-called out-and-back experiments) which are less sensitive, but due to the better relaxation properties of the protein the spectra may none-the-less improve.
Field strength
In principle it is good to move to higher fields (750 MHz or more) with larger proteins, since it is then possible to use TROSY techniques and the greater resolution helps when the degree of overlap is large. However, it is worth noting that the CBCA(CO)NNH or HN(CO)CA are likely to decrease in quality due to the increased effect of the carbonyl chemical shift anisotropy. So even for proteins of 200 or more amino acids it may prove better to record 3D data sets at 600 MHz.
Top
Visualising 3D Spectra
3D experiments are generally based upon 2D experiments and so the easiest way to think of a 3D is of a 2D which is then extended into a third dimension. Take, for instance, an
HNCO. It is based upon a 2D
HSQC (a), so the x and y axes are 1H and 15N, respectively. This is now extended into a third dimension which is a 13C dimension. So the HSQC peaks will now not just lie in one plane, but they will be lifted up into the third dimension and lie at the 13C ppm value of the CO group preceeding the NH group (b and c).
It is now possible to look at the 3D spectrum from various different angles and each time see a different plane. The 1H dimension is generally left in the x-dimension and in most cases the 13C dimension is viewed along the y-axis, leaving 15N to form the z-plane. So essentially you end up looking at a 1H-13C 2D spectrum at varying places along the 15N dimension (d and e).
Most spectra used for triple resonance backbone assignment have a 1H, 15N and 13C dimension each. Several other types of spectra, most notably 3D NOESY spectra and HCCH-TOCSY/COSY spectra have two 1H dimensions and one 15N or 13C dimension. In this case, the two 1H dimensions are viewed in x and y and the 15N or 13C is left in the z-plane.
A common way of visualising 3D spectra is as so-called
Strips. Consider, for instance, a particular z-plane of a 3D HNCO spectrum (a or b): you will probably end up with only one peak in that z-plane. (Even if there are peaks with a similar 15N ppm value, they will lie just above or just below the actual plane you are currently looking at.) Therefore there is not much point in looking at the complete 1H width of the spectrum and instead you can trim the area you look at to the 1H region just surrounding your peak (c and d). This way you end up with a
Strip. It corresponds to a particular 1H and 15N part of the spectrum, but shows the complete 13C width. Thus, if you start with an HSQC where each peak is defined by a 1H and 15N value, you can then pick out strips for each HSQC peak and then lay them next to one another for easy comparison (e). For protein backbone assignment this is particularly useful.
Top
Triple Resonance Backbone Assignment
Standard triple resonance backbone assignment of proteins is based on the
CBCANNH and
CBCA(CO)NNH spectra. The idea is that the CBCANNH correlates each NH group with the Cα and Cβ chemical shifts of its own residue (strongly) and of the residue preceding (weakly). The CBCA(CO)NNH only correlates the NH group to the preceding Cα and Cβ chemical shifts. The Figure below shows how this can be used to link one NH group to the next into a long chain.
In practise, using the CBCANH and CBCA(CO)NH spectra this looks like this (Cαs are shown in dark blue, Cβs in light blue):
Alternatively, some software packages (such as CCPNmr Analysis) allow you to superimpose the two spectra and then your strips will look like this:
The Cα and Cβ chemical shifts adopt values characteristic of the amino acid type. Some of these, such as Alanine, Serine, Threonine and Glycine are very easy to spot as their Cβ chemical shifts are very different to those of the other amino acids (and in the case of Glycine there is no Cb). Valine, Isoleucine and Proline are also likely to stand out by the fact that they have lower than normal Cα chemical shifts. Once a chain of NH groups with their corresponding Cα and Cβ chemical shifts has been built, then the identification of some of the amino acid types makes it possible to match this string to the sequence. E.g. a string of shifts may have been found that corresponds to xxxSxxAx - if this sequence only appears once in the sequence of the protein in question, then sequence-specific assignment can be made.
In some cases, in particular if your protein is fairly large (>200 residues, say), you may find that the quality of the CBCANNH and CBCA(CO)NNH spectra are not very good. The Cβ resonances may, for example, not be visible above the noise level. In this case it is possible to use the Cα and C' chemical shifts rather than the Cα and Cβ chemical shifts, as those which you use to walk from one residue to the next. The
HNCA and
HN(CO)CA experiments give you the same information as the CBCANNH and CBCA(CO)NNH spectra, except without the Cβ resonances. To complement this, you can then record the
HNCO and
HN(CA)CO experiments. These link each NH(i) group with the C'(i-1) (HNCO) or with C'(i) and C'(i-1) (HN(CA)CO). The residues are now linked up in the following manner:
The advantage of using the HNCO and HNCA-based spectra is that they are more sensitive than the CBCANNH-type and thus the spectral quality should improve. The disadvantage is that the Cα and C' chemical shifts provide less information about the amino acid type than the Cβ chemical shift and are less disperse.
Top
Triple Resonance Side Chain Assignment
Various methods and spectra are available - it depends a bit on the size of the protein and the amount of spectrometer time available as to what spectra are used.
A straight forward method is to begin with a set of
HBHA(CO)ONH,
HCC(CO)NNH and
CC(CO)NNH spectra. These will provide the hydrogen and carbon side-chain chemical shifts for the residue preceding each NH group. For longer side chains not all peaks may neccessarily be visible, so that this may not be sufficient. In some cases it may also be difficult to distinguish between say an Hβ and an Hγ shift. Furthermore the connectivity of which hydrogen is attached to which carbon is also not provided. This is, for instance, relevant in the case of Valines where there are two methyl groups: it may be possible to identify both methyl carbon and both methyl hydrogen chemical shifts, but it will not be known which is bound to which.
The most useful spectrum for side-chain assignment is an
HCCH-TOCSY spectrum along with the
HCCH-COSY spectrum. The HCCH-TOCSY will at any one carbon position show in one dimension the chemical shift of the hydrogen which is attached to the carbon and in another the other hydrogens belonging to that side chain. There is thus a huge amount of information in this spectrum and for large proteins it may become rather crowded. The HCCH-COSY is similar, except that instead of seeing all hydrogens belonging to a certain side-chain in the third dimension, you only see those which are attached to neighbouring carbon atoms. The following figure shows the strips you should be able to see for a Valine residue in the HCCH-TOCSY and HCCH-COSY spectra.
The general principle behind using the HCCH-TOCSY spectrum is as follows: Using your known Cα and Cβ chemical shifts from the backbone assignment, you go to these points in the spectrum. You will then immediately get the Hα and Hβ chemical shifts by finding strips at each carbon shift which have peaks at the same hydrogen ppm values. You should also be able to see further peaks for the Hγ and Hδ atoms (if present in that particular amino acid type). By navigating to these new hydrogen shifts you should in turn be able to identify the shifts of the carbons they are attached to.
Top
Double Resonance Backbone Assignment
For smaller proteins, it is possible to do the backbone assignment using just 15N-labelled protein. The spectra used for this are the
15N-NOESY-HSQC and the
15N-TOCSY-HSQC. The 15N-NOESY-HSQC will show for each NH group all 1H resonances which are within about 5-7Å of the NH hydrogen. Assignment is done on the assumption that the two neighbouring NH groups are always visible. Thus two NH groups can be linked because they each have an NOE to the other NH group.
Note that you always end up with a square motif between between strips which are linked by an NOE: each strip has an NOE to the diagonal peak of the other strip.
Helical sections are generally easier to assign, as NOEs from NH(i) are visible not only to NH(i±1), but also to NH(i±2) and sometimes NH(i±3).
β-sheet structures include short NH-NH distances between the strands. This means that in addition to the NH(i±1) NOEs, a strong cross-strand NOE is also observed.
Having a rough idea of the secondary structure and topology of the protein can thus significantly aid backbone assignment using double resonance spectra only.
Further help with assignment is provided by the 15N-TOCSY-HSQC. This should show links from the backbone NH group to all side-chain hydrogens of that residue. Using this spectrum the amino acid type can be identified or narrowed down significantly.
The side-chain NOEs from the 15N-NOESY-HSQC can also be useful during the assignment process, as NH(i)-Hα(i-1) are generally very strong, in particular in β-sheet sections.
Top
Double Resonance Side Chain Assignment
Assignment of side-chain hydrogen atoms using double-resonance spectra, only, may not always be possible with complete unambiguity. The easiest spectrum to use is the
15N-TOCSY-HSQC, as this should show all side-chain hydrogen resonances for any given NH group. However, in many cases not all side-chain resonances may be visible, or for certain amino acid types it may not be possible to assign the resonances to particular side-chain groups. In particular it may in some cases be difficult to distinguish between Hβ and Hγ atoms and it may not always be possible to work out whether mythylene groups are degenerate or not.
